Rabbit anti bcl 2

For immunohistochemical staining, the primary antibodies (rabbit anti–Bcl-2,1:1000, Biorbyt, Cambridge, UK; rabbit anti–Caspase-3, 1:1000, CST, Boston, MA, USA) incubated with the sections overnight at 4 °C and visualized by incubating 0.05% 3,3–diaminobenzidine tetrahydrochloride (DAB, Sigma, St. Louis, MO, USA) and 0.003% hydrogen peroxide. A total of 25 fields were randomly selected in each sample. The integrated optical densities (IODs) of positive cells were measured by using Image–Pro Plus software.

The proteins (n = 5) of bursa were extracted with RIPA lysis buffer and determined concentration with the bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China). Then, the equal amount of protein in each group was added to the SDS–polyacrylamide gel and transferred onto PVDF membranes and blocked for 1 h using 5% skimmed milk. Subsequently, the primary antibodies, including goat anti–Bax (1:1000, Biorbyt, Cambridge, UK), rabbit anti–Bcl–2 (1:1000, Biorbyt, Cambridge, UK), rabbit anti–Caspase–3 (1:1000, CST, Boston, MA, USA), rabbit anti–Nrf2 (1:1000, Proteintech Group, Inc., Wuhan, China), rabbit anti–HO-1 (1:1000, BIOSS, Beijing, China), rabbit anti–p-iκb (1:1000, Abcam, Cambridge, UK), rabbit anti–p-p65 (1:1000, Abcam, Cambridge, UK) or rabbit anti–β–actin (1:4000; Co Win Biotech Co., Inc., Beijing, China), incubated with the membranes overnight at 4 °C. Then, the membranes were washed with TBST and incubated with horseradish peroxidase–conjugated goat anti–mouse/rabbit IgG (1:8000; Co Win Biotech Co., Inc., Beijing, China) for 2 h. The target bands obtained in the blots were scanned and measured using ImageJ 4.0.2 software (Scion Corp., Frederick, MD, USA) The data were expressed as the IOD of the target bands and compared to the corresponding β–actin values. Repeat the test three times for each sample.